Phenylboronic Acid-Functionalized Micelles Dual-Targeting Boronic Acid Transporter and Polysaccharides for siRNA Delivery into Brown Algae.

Brown algae play essential roles ecologically, practically, and evolutionarily because they maintain coastal areas, capture carbon dioxide, and produce valuable chemicals such as therapeutic drugs. To unlock their full potential, understanding the unique molecular biology of brown algae is imperative. Genetic engineering tools that regulate homeostasis in brown algae are essential for determining their biological mechanisms in detail. However, few methodologies have been developed to control gene expression due to the robust structural barriers of brown algae. To address this issue, we designed peptide-based, small interfering RNA (siRNA)-loaded micelles decorated with phenylboronic acid (PBA) ligands. The PBA ligands facilitated the cellular uptake of the micelles into a model brown alga, Ectocarpus siliculosus (E. Siliculosus), through chemical interaction with polysaccharides in the cell wall and biological recognition by boronic acid transporters on the plasma membrane. The micelles, featuring "kill two birds with one stone" ligands, effectively induced gene silencing related to auxin biosynthesis. As a result, the growth of E. siliculosus was temporarily inhibited without persistent genome editing. This study demonstrated the potential for exploring the characteristics of brown algae through a simple yet effective approach and presented a feasible system for delivering siRNA in brown algae.

Plant Mitochondrial-Targeted Gene Delivery by Peptide/DNA Micelles Quantitatively Surface-Modified with Mitochondrial Targeting and Membrane-Penetrating Peptides.

Plant mitochondria play essential roles in metabolism and respiration. Recently, there has been growing interest in mitochondrial transformation for developing crops with commercially valuable traits, such as resistance to environmental stress and shorter fallow periods. Mitochondrial targeting and cell membrane penetration functions are crucial for improving the gene delivery efficiency of mitochondrial transformation. Here, we developed a peptide-based carrier, referred to as Cytcox/KAibA-Mic, that contains multifunctional peptides for efficient transfection into plant mitochondria. We quantified the mitochondrial targeting and cell membrane-penetrating peptide modification rates to control their functions. The modification rates were easily determined from high-performance liquid chromatography chromatograms. Additionally, the gene carrier size remained constant even when the mitochondrial targeting peptide modification rate was altered. Using this gene carrier, we can quantitatively investigate the relationships between various peptide modifications and transfection efficiency and optimize the gene carrier conditions for mitochondrial transfection.

Organelle-targeted gene delivery in plants by nanomaterials.

Genetic engineering of plants has revolutionized agriculture and has had a significant impact on our everyday life. It has allowed for the production of crops with longer shelf lives, enhanced yields and resistance to pests and disease. The application of nanomaterials in plant genetic engineering has further augmented these programs with higher delivery efficiencies, biocompatibility and the potential for plant regeneration. In particular, subcellular targeting using nanomaterials has recently become possible with the cutting-edge developments within nanomaterials, but remains challenging despite the promise in organellar engineering for the introduction of useful traits and the elucidation of subcellular interactions. This feature article provides an overview of nanomaterial delivery within plants and highlights the application of recent progress in nanomaterials for subcellular organelle-targeted delivery.

Relaxation of the Plant Cell Wall Barrier via Zwitterionic Liquid Pretreatment for Micelle-Complex-Mediated DNA Delivery to Specific Plant Organelles.

Targeted delivery of genes to specific plant organelles is a key challenge for fundamental plant science, plant bioengineering, and agronomic applications. Nanoscale carriers have attracted interest as a promising tool for organelle-targeted DNA delivery in plants. However, nanocarrier-mediated DNA delivery in plants is severely hampered by the barrier of the plant cell wall, resulting in insufficient delivery efficiency. Herein, we propose a unique strategy that synergistically combines a cell wall-loosening zwitterionic liquid (ZIL) with a peptide-displaying micelle complex for organelle-specific DNA delivery in plants. We demonstrated that ZIL pretreatment can enhance cell wall permeability without cytotoxicity, allowing micelle complexes to translocate across the cell wall and carry DNA cargo into specific plant organelles, such as nuclei and chloroplasts, with significantly augmented efficiency. Our work offers a novel concept to overcome the plant cell wall barrier for nanocarrier-mediated cargo delivery to specific organelles in living plants.

A Synthetic Multidomain Peptide That Drives a Macropinocytosis-Like Mechanism for Cytosolic Transport of Exogenous Proteins into Plants.

Direct delivery of proteins into plants represents a promising alternative to conventional gene delivery for probing and modulating cellular functions without the risk of random integration of transgenes into the host genome. This remains challenging, however, because of the lack of a protein delivery tool applicable to diverse plant species and the limited information about the entry mechanisms of exogenous proteins in plant cells. Here, we present the synthetic multidomain peptide (named dTat-Sar-EED4) for cytosolic protein delivery in various plant species via simple peptide-protein coincubation. dTat-Sar-EED4 enabled the cytosolic delivery of an active enzyme with up to ∼20-fold greater efficiency than previously described cell-penetrating peptides in several model plant systems. Our analyses using pharmacological inhibitors and transmission electron microscopy revealed that dTat-Sar-EED4 triggered a unique endocytic mechanism for cargo protein internalization. This endocytic mechanism shares several features with macropinocytosis, including the dependency of actin polymerization, sensitivity to phosphatidylinositol-3 kinase activity, and formation of membrane protrusions and large intracellular vesicles (>200 nm in diameter), even though macropinocytosis has not been identified to date in plants. Our study thus presents a robust molecular tool that can induce a unique cellular uptake mechanism for the efficient transport of bioactive proteins into plants.

Fusion Peptide-Based Biomacromolecule Delivery System for Plant Cells.

The introduction of DNA, RNA, and proteins into plant cells has become important in plant science with the recent development of innovative technologies such as genome editing. As a new method for the delivery of such biomacromolecules, fusion peptides, which have multiple functional domains, have been developed. The functional domains include cell-penetrating peptides for crossing cell membranes, polycationic peptides for biomacromolecule binding, and organelle-targeting peptides. The fusion peptide-based macromolecule delivery system enables the efficient introduction of DNA, RNA, and proteins, which are much larger in size than the peptide, into plant cells while retaining the activity of the biomacromolecules. Compared to pre-existing delivery methods, this system has advantages in that it does not require any special equipment and can be performed easily and quickly on a wide variety of plants. Furthermore, as a characteristic feature of the fusion peptide system, the application of organelle-targeting peptides to fusion peptides allows selective delivery of biomacromolecules to chloroplasts or mitochondria. Here, we provide a representative method of the fusion peptide-based biomacromolecule delivery system and an example of the results of biomacromolecule delivery as promising new tools for plant biology and biotechnology.

Endosome-escaping micelle complexes dually equipped with cell-penetrating and endosome-disrupting peptides for efficient DNA delivery into intact plants.

The delivery of DNA to plants is crucial for enhancing their ability to produce valuable compounds and adapt to climate change. Peptides can provide a versatile tool for delivering DNA to a specific target organelle in various plant species without the use of specialized equipment. However, peptide-mediated DNA delivery suffers from endosomal entrapment and subsequent vacuolar degradation of the DNA cargo, which leads to poor transfection efficiency. To overcome the lack of a reliable approach for bypassing vacuolar degradation in plants, we herein present an endosome-escaping micelle. The micelle surface is dually modified with cell-penetrating (CPP) and endosome-disrupting peptides (EDP) and the core is composed of plasmid DNA condensed with cationic peptides. Due to the functions of CPP and EDP, the dual peptide-modified micelles efficiently undergo endocytic internalization and escape from endosomes to the cytosol, thereby achieving significantly enhanced transfection of intact plants with negligible cytotoxicity. The present study offers a robust strategy for efficient intracellular DNA delivery to plants without vacuolar degradation, and can facilitate plant bioengineering for diverse biotechnological applications.

Peptide-Mediated Gene Transfer into Marine Purple Photosynthetic Bacteria.

Use of photosynthetic organisms is one of the sustainable ways to produce high-value products. Marine purple photosynthetic bacteria are one of the research focuses as microbial production hosts. Genetic transformation is indispensable as a biotechnology technique. However, only conjugation has been determined to be an applicable method for the transformation of marine purple photosynthetic bacteria so far. In this study, for the first time, a dual peptide-based transformation method combining cell penetrating peptide (CPP), cationic peptide and Tat-derived peptide (dTat-Sar-EED) (containing D-amino acids of Tat and endosomal escape domain (EED) connected by sarcosine linkers) successfully delivered plasmid DNA into Rhodovulum sulfidophilum, a marine purple photosynthetic bacterium. The plasmid delivery efficiency was greatly improved by dTat-Sar-EED. The concentrations of dTat-Sar-EED, cell growth stage and recovery duration affected the efficiency of plasmid DNA delivery. The delivery was inhibited at 4 °C and by A22, which is an inhibitor of the actin homolog MreB. This suggests that the plasmid DNA delivery occurred via MreB-mediated energy dependent process. Additionally, this peptide-mediated delivery method was also applicable for E. coli cells. Thus, a wide range of bacteria could be genetically transformed by using this novel peptide-based transformation method.

Dual Peptide-Based Gene Delivery System for the Efficient Transfection of Plant Callus Cells.

Owing to their diverse functions and tunable physicochemical properties, peptides are promising alternatives to the conventional gene delivery tools that are available for plant systems. However, peptide-mediated gene delivery is limited by low transfection efficiency in plants because of the insufficient cytosolic translocation of DNA cargo. Here, we report a dual peptide-based gene delivery system for the efficient transfection of plant callus cells. This system is based on the combination of an artificial peptide composed of cationic cell-penetrating and hydrophobic endosomal escape domains with a gene carrier peptide composed of amphiphilic cell-penetrating and cationic DNA-binding domains. Cellular internalization and transfection studies revealed that this dual peptide-based system enables more efficient transfection of callus cells than does a carrier peptide alone by enhancing the endocytic uptake and subsequent cytosolic translocation of a carrier peptide/DNA complex. The present strategy will expand the utility of peptide-mediated plant gene delivery for a wide range of applications and basic research.

Zwitterionic Polypeptides: Chemoenzymatic Synthesis and Loosening Function for Cellulose Crystals.

A polypeptide with a GlyHisGly repeating sequence containing zwitterionic structures that effectively interact with cellulose was synthesized for dissociation of cellulose crystals. Polypeptide with the GlyHisGly sequence was synthesized by chemoenzymatic polymerization and postfunctionalization of the His residues was performed to afford imidazolium butyrate on the side chains. The resulting zwitterionic polypeptide effectively dissociated bundles of tunicate cellulose nanocrystals, even when the conditions were mild and the concentration of the polypeptide was as low as 1-2 mg mL-1. Polypeptide treatment also affected the morphology of the cell walls in cultured plant cells, and the cellulose microfibril networks and amorphous polysaccharide layer were dissociated according to atomic force microscopy (AFM). The zwitterionic polypeptide treatment did not change the crystal structure of the cellulose nanocrystals. Analysis of the mechanical properties of the cellulose nanocrystals by force curve measurements using AFM revealed that the elastic modulus of the cellulose nanocrystals increased after treatment with the zwitterionic polypeptide, indicating that the amorphous part of the cellulose nanocrystals was removed by interactions with the polypeptide. At a concentration of the polypeptide that enabled the dissociation of the cellulose network, the zwitterionic polypeptide showed negligible cytotoxicity to the plant cells. The mild and noncytotoxic technique for loosening cellulose microfibrils/nanocrystals that was developed in this study has tremendous significance for the modification of cellulose in terms of polymer chemistry, material science, and plant biotechnology.

Construction of a Quadrangular Tetramer and a Cage-Like Hexamer from Three-Helix Bundle-Linked Fusion Proteins.

Self-assembled protein nanostructures have gained interest, owing to their potential applications in biomaterials; however, successful design and construction of protein nanostructures are limited. Herein, we constructed fusion protein 1 by linking the C-terminus of a dimerization domain and the N-terminus of another dimerization domain with a three-helix bundle protein, where it self-assembled mainly into tetramers. By replacing the C-terminal dimerization domain of 1 with a trimerization domain (fusion protein 2), hexamers were mainly obtained. According to ab initio structural models reconstructed from the small-angle X-ray scattering data, the tetramer of 1 and hexamer of 2 adopted quadrangle and cage-like structures, respectively, although they were combinations of different conformations. High-speed atomic force microscopy observations indicated that the tetramer and hexamer exhibit conformational dynamics. These results show that the present method utilizing three-helix bundle-linked fusion proteins is useful in the construction of protein nanostructures.

Block Copolymer/Plasmid DNA Micelles Postmodified with Functional Peptides via Thiol-Maleimide Conjugation for Efficient Gene Delivery into Plants.

Introducing exogenous genes into plant cells is essential for a wide range of applications in agriculture and plant biotechnology fields. Cationic peptide carriers with cell-penetrating and DNA-binding domains successfully deliver exogenous genes into plants. However, their cell-penetrating activity may be attenuated by undesired electrostatic interactions between the cell-penetrating peptide (CPP) domain and DNA cargo, resulting in limited gene delivery efficiency. Here, we developed the block copolymer maleimide-conjugated tetra(ethylene glycol) and poly(l-lysine) (MAL-TEG-PLL). Through electrostatic interactions with plasmid DNA (pDNA), MAL-TEG-PLL formed a micelle that presented maleimide groups on its surface. The micelle enabled postmodification with cysteine-containing functional peptides, including a CPP (BP100-Cys) and nuclear localization signal (Cys-NLS) via thiol-maleimide conjugation, thereby avoiding undesired interactions. According to a comparison of gene delivery efficiencies among the peptide-postmodified micelles, the amount of BP100-Cys on the micelle surface was key for efficient gene delivery. The BP100-postmodified micelle showed more efficient delivery compared with that of the BP100-premodified micelle. Thus, postmodification of polymeric micelles with functional peptides opens the door to designing highly efficient plant gene delivery systems.

Enhanced stability of Cu(2+)-ATCUN complexes under physiologically relevant conditions by insertion of structurally bulky and hydrophobic amino acid residues into the ATCUN motif.

Copper complexes formed by an amino terminal Cu(2+)- and Ni(2+)-binding (ATCUN) motif have attracted attention as metallodrug candidates that cleave DNA or RNA and inactivate enzymes. Although the stability of the Cu(2+)-ATCUN complex under physiologically relevant conditions is a key factor for medical applications, it has remained unclear. Here we prepared a series of ATCUN peptides by inserting various amino acid residues into positions 1 and 2, and investigated the stability of the Cu(2+)-ATCUN complexes in aqueous solution, blood plasma, and living animals. Systematic pH titration showed that the low basicity of the N-terminal amine of the peptide stabilized the Cu(2+)-ATCUN complex in aqueous solution. Interestingly, the stability of (64)Cu-labeled ATCUN complexes in blood plasma was significantly enhanced by the structural bulkiness and hydrophobicity of the amino acid residues at positions 1 and 2. To validate the in vivo stability, six ATCUN motifs (YYH, VVH, NNH, TTH, GGH, and DDH) were conjugated to a tumor-targeting peptide, octreotide (Oct). The stability of the (64)Cu-ATCUN-Oct complexes in blood plasma showed a similar trend to that of the (64)Cu-ATCUN complexes. The (64)Cu-YYH-Oct complex exhibited the highest stability in blood plasma. According to the positron emission tomography and competitive blocking studies of a tumor-bearing mouse model, (64)Cu-YYH-Oct specifically accumulated in tumors, suggesting that the complex was sufficiently stable to reach its target in vivo. The results show that the structural bulkiness and hydrophobicity of the residues at positions 1 and 2 are key parameters for designing metallodrugs on the basis of the Cu(2+)-ATCUN complex.

Domain-swapped cytochrome cb562 dimer and its nanocage encapsulating a Zn-SO4 cluster in the internal cavity.

Protein nanostructures have been gaining in interest, along with developments in new methods for construction of novel nanostructures. We have previously shown that c-type cytochromes and myoglobin form oligomers by domain swapping. Herein, we show that a four-helix bundle protein cyt cb562, with the cyt b562 heme attached to the protein moiety by two Cys residues insertion, forms a domain-swapped dimer. Dimeric cyt cb562 did not dissociate to monomers at 4 °C, whereas dimeric cyt b562 dissociated under the same conditions, showing that heme attachment to the protein moiety stabilizes the domain-swapped structure. According to X-ray crystallographic analysis of dimeric cyt cb562, the two helices in the N-terminal region of one protomer interacted with the other two helices in the C-terminal region of the other protomer, where Lys51-Asp54 served as a hinge loop. The heme coordination structure of the dimer was similar to that of the monomer. In the crystal, three domain-swapped cyt cb562 dimers formed a unique cage structure with a Zn-SO4 cluster inside the cavity. The Zn-SO4 cluster consisted of fifteen Zn2+ and seven SO42- ions, whereas six additional Zn2+ ions were detected inside the cavity. The cage structure was stabilized by coordination of the amino acid side chains of the dimers to the Zn2+ ions and connection of two four-helix bundle units through the conformation-adjustable hinge loop. These results show that domain swapping can be applied in the construction of unique protein nanostructures.

[Development of new peptide-based chelating agents for site-specific radiolabeling with 64Cu].

Radiolabeled monoclonal antibodies (mAbs) are very useful molecular probes for nuclear imaging technique due to their high-target specificity and high-stability in the bloodstream. MAbs are generally labeled by indirect method using the combination of relatively long-lived metallic radionuclides (including (64)Cu, (89)Zr, and (111)In) and bifunctional chelating agents which are capable of binding both antibodies and radio metals. The indirect radiolabeling method has some advantages such as high labeling efficiency and long-term retention ability within their target cells. However, this conventional labeling method can potentially lead to low-target affinity of the mAb probes, because of the non-site-specific introduction of the bifunctional chelators into the active site of the mAbs. To overcome the shortcoming, we proposed a new direct labeling method utilizing fusion proteins comprising mAbs linked to metal binding peptides at the N- or C-terminus. In this study, we synthesized new peptide derivatives possessing an N-terminal tripeptide sequence (Xaa-Yaa-His) called the amino terminal Cu(2+)- and Ni(2+)-binding (ATCUN) motif as (64)Cu binding peptides for the proposed labeling method. Moreover, we studied the stability constants of Cu(2+)-ATCUN peptide complexes by pH titration. From these studies, we found that a low basicity of the N-terminal amine in the peptide resulted in a high stability constant of the complex. This finding may provide valuable guidelines in designing the ATCUN peptide with high-binding affinity toward (64)Cu.